Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, in a large and spacious phagolysosome-like parasitophorous vacuole (PV). Recruitment of membrane during PV biogenesis is a complex process modulated by both host and bacterial factors. Coxiella encodes a specialized Dot/Icm type IVB secretion system (T4BSS) that secretes proteins with effector functions directly into the host cell cytosol. Effector proteins are predicted to modulate an array of host cell processes, such as vesicular trafficking, that promote pathogen growth. Coxiella Dot/Icm function was initially studied using Legionella pneumophila as surrogate host. However, by using new gene inactivation technologies developed in our laboratory, we have recently confirmed that a functional T4BSS is required for productive infection of human macrophages by Coxiella. Furthermore, we have verified Dot/Icm-dependent secretion by different strains of Coxiella of over 40 proteins. Coxiella must co-opt vesicular trafficking pathways to promote PV development. We are currently elucidating the activities of five effector proteins that traffic to the PV membrane termed CvpA (Coxiella vacuolar protein A), CvpB, CvpC, CvpD, and CvpE that are speculated to modulate membrane fusion events. Mutants in individual cvp genes all display significant defects in replication and PV development. Particular insight into the function of CvpA has been gained by showing the protein subverts clathrin-coated vesicle trafficking. Additional cell biologic studies have increased our knowledge of Coxiella parasitism. We demonstrated that Coxiella infection expands the host endosomal system to promote PV formation. We showed that high levels of PV cholesterol are toxic for Coxiella, suggesting targeting of host cholesterol metabolism might prove clinically useful in controlling infection. Unlike previous studies, we demonstrated primary murine bone marrow derived-macrophages support growth of the workhorse, avirulent Nine Miles phase II (NMII) strain if macrophages are differentiated with macrophage colony-stimulating factor. This allows infection of macrophages from knockout mice by widely-used NMII to model Q fever pathogenesis. Finally, we showed that vasodilator-stimulated phosphoprotein, a protein kinase A substrate and actin regulatory protein, is critical for PV expansion. Regulation of the Coxiella T4BSS is poorly defined. IcmS is a predicted cytoplasmic adapter protein that facilitates translocation of certain T4BSS effectors by binding an internal signal sequence(s). We examined the function of Coxiella IcmS by generating an icmS deletion mutant. The Coxiella icmS mutant grows normally in axenic media while having a pronounced growth defect in host cells that is rescued with a single chromosomal copy of icmS. Optimal secretion of individual substrates is either IcmS-dependent or independent. Additionally, a subset of substrates display hyper-secretion in the Coxiella icmS mutant, suggesting IcmS may also suppress secretion of some Dot/Icm substrates. Thus, regulation by IcmS appears complex with the growth defect of the Coxiella icmS mutant potentially explained by both deficient and aberrant secretion of effector proteins. A hallmark of Coxiella is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCV are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. Although the developmental cycle is considered fundamental to Coxiella virulence, the molecular biology of this process is poorly understood. Fortunately, we discovered that Coxiella developmental transitions and viability in the synthetic medium ACCM-D mimic host cell-cultivated organisms. Axenic cultivation of Coxiella in ACCM-D, along with new methods for genetic manipulation, now provides powerful tools to investigate the molecular basis and biological relevance of Coxiella biphasic development. Ultrastructural studies show marked differences in the cell envelope between cell variants, but little is known about biochemical differences between SCV and LCV that confer their distinct biological and physical properties. We analyzed the lipid composition of Coxiella after 4 (LCV), 7 (intermediate forms) and 14 (SCV) days of growth in synthetic medium, using thin layer chromatography and mass spectrometry. Similar to Escherichia coli, Coxiella contains cardiolipin, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE), with some PE in an unusual plasmalogen form. PE and PG are present in lower quantities in the SCV relative to the LCV. However, three additional major lipid species are present in higher quantities in the SCV: lyso-phosphatidylethanolamine, a breakdown product of PE; glycerophospho-N-acyl-ethanolamine, a lipid previously not found in bacteria; and free fatty acids, which are normally toxic for bacteria. Mutational analysis indicates that these three lipids are generated via the activity of a Coxiella outer membrane phospholipase A homolog (CBU0489). A cbu0489 mutant exhibits a significant growth defect in THP-1 macrophage-like cells, suggesting developmentally regulated lipid synthesis is required for optimal intracellular growth and could contribute to the distinct properties of LCV and SCV. To further identify genetic determinants of LCV to SCV transition, we profiled the Coxiella transcriptome by microarray at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV) and 21 (late SCV) days post-infection (dpi) of Vero epithelial cells. Transcriptional signatures of SCV are up-regulation of genes involved in oxidative stress responses, arginine metabolism, and cell wall remodeling. Genes down-regulated in SCV are primarily associated with intermediary metabolism. A striking transcriptional signature of the SCV is induction (10-fold) of five genes encoding predicted L,D transpeptidases that catalyze beta-lactam resistant 3-3 peptide crosslinks typically found in the peptidoglycan (PG) of stationary phase bacteria. Cryo-electron microscopy reveals an unusually thick and dense periplasmic layer specific to SCV, suggestive of PG, whereas the periplasm and inner and outer membranes of LCV exhibits a more typical gram-negative appearance. Muropeptide analysis of Coxiella PG shows an increasing percentage of 3-3 cross-links as LCV transition to SCV raising the possibility that the Coxiella L,D transpeptidases may be important in cross-linking the PG of SCV. Collectively, these results indicate the SCV produces a unique transcriptome with a major subset of these genes directed towards remodeling a PG layer that may contribute to environmental resistance by Coxiella.